- Email: [email protected]
Studies on the antifungal activity of biotemplated gold nanoparticles over Candida albicans
Accepted Manuscript Title: Studies on the antifungal activity of biotemplated gold nanoparticles over Candida albicans Authors: Nidhin M, Saneha D, Sandeep Hans, Anitha Varghese, Zeeshan Fatima, Saif Hameed PII: DOI: Article Number:
S0025-5408(18)32533-9 https://doi.org/10.1016/j.materresbull.2019.110563 110563
Reference:
MRB 110563
To appear in:
MRB
Received date: Revised date: Accepted date:
17 September 2018 20 July 2019 25 July 2019
Please cite this article as: M N, D S, Hans S, Varghese A, Fatima Z, Hameed S, Studies on the antifungal activity of biotemplated gold nanoparticles over Candida albicans, Materials Research Bulletin (2019), https://doi.org/10.1016/j.materresbull.2019.110563 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Studies on the antifungal activity of biotemplated gold nanoparticles over Candida albicans Nidhin Ma,*, Saneha Db, Sandeep Hansc, Anitha Varghesea, Zeeshan Fatimac, Saif Hameedc,* a
Department of Chemistry, CHRIST (Deemed to be University), Bengaluru, Karnataka 560029, India Department of Chemistry, Amity School of Applied Sciences, Amity University Haryana Amity Education Valley, Gurgaon, Haryana 122413,India c Amity Institute of Biotechnology, Amity University Haryana, Amity Education Valley, Gurgaon, Haryana 122413,India b
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*Corresponding Authors: Nidhin M, Department of Chemistry, CHRIST (Deemed to be University), Bengaluru, Karnataka 560029
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Email: [email protected] , [email protected]
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Email : [email protected]
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Dr. Saif Hameed, Amity Institute of Biotechnology, Amity University Haryana, Gurgaon-
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Graphical abstract
Highlights The synthesis of gold nanoparticles and their biomedical applications are always a very hot topic in the scientific community. This paper highlights the synthesis of gold nanoparticles of uniform size around 5nm with spherical shape using starch as a biotemplate. X-Ray diffraction, UV-visible Spectroscopy, Transmission Electron Microscopy and Atomic Force Microscopy were employed to characterize or analyzed the synthesized gold nanoparticles.
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The biomedical applications of the synthesized gold nanoparticles were carried out against most prevalent human fungal pathogen, Candida albicans. Broth micro dilution assay to determine Minimum inhibitory concentration (MIC) was used to find out the concentration of
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gold nanoparticles at which it inhibits the growth of fungal cells. We observed that 0. 5 mM
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concentration was sufficient to inhibit the growth of cells in which was also confirmed by
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spot assay.
ABSTRACT Green synthesis and applications of gold nanoparticles are more fascinating research area due to their unique optical properties and high X-ray attenuation power. In this study, we have synthesized gold nanoparticles of uniform size (5 nm) with spherical shape. UV-visible spectroscopy, Transmission Electron Microscopy and Atomic Force Microscopy were employed to characterize the synthesized gold nanoparticles. The biomedical applications of the synthesized gold nanoparticles were carried out against most prevalent human fungal pathogen, Candida albicans. Broth micro dilution assay was used to determine minimum inhibitory concentration (MIC). We observed that 0. 5 mM concentration was effective in inhibiting the growth of fungal cells which was later confirmed by spot assay. Key Words: Gold Nanoparticles, Antifungal Activity, Candida albicans, Micro Dilution
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Assay, Green Synthesis
1. Introduction
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In the modern era of material science, nanoparticles have become the centre of attraction to the scientific community. In current years, nanotechnology is one of the most researched areas due to their noticeable performance in electronics, optics and photonics. Nanoparticles are the fundamental structures of nanotechnology.[1] Nanoscience and technology is an interdisciplinary broad area of research and development activity that has been growing dynamically worldwide in the past few years. Nanoparticles are the simplest form of structures with the range of 1-100 nm [2]. Nanoparticles have different physical and chemical properties such as higher surface area, mechanical strength and high reactivity. Gold nanoparticles are unique in optical property as gold is yellow in shading, strong in state where as gold nanoparticles are wine red shading arrangement against oxidant [3]. Gold nanoparticles display different sizes extending from 1 nm to 8 μm and they likewise show distinctive shapes, such as round, sub-octahedral, octahedral, decahedral, sporadic shape, tetrahedral, hexagonal platelets and nanorods. Among all these shapes, circular molded nanoparticles are most steady and show alluring optical properties when contrasted with the triangular formed nanoparticles [4]. Conventional physical and chemical methods are used to prepare metal nanoparticles from toxic chemicals [5]. Moreover, these methods are very expensive and not environmentally friendly [6]. Green synthesis of gold nanoparticles using various templates such as polysaccharides, fungi and plant extracts are found to be environmentally benign [7]. These attractive green strategies are free from toxic chemicals and toxic materials. Gold nanoparticle synthesized from bio templates offers a route for large scale production of different metallic nanoparticles [8].
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Bio templated gold nanoparticles are utilized to detect cancer cells. Increased surface area of gold nanoparticles in solution which contribute to their enhanced physio-chemical properties which are useful in a variety of fields such as antimicrobial agents [9], bio-molecular detection, diagnostics, catalysis, biomedical and bio sensing devices. Gold nanoparticles are utilized as efficient materials for water purification [10]. These are also used in interface resistors, conductors and different components of an electronic chip. In photodynamic treatment, when light is connected to a tumor containing gold nanoparticles, the particles quickly warm up, executing tumor cells [11]. Gold nanoparticles are used as substrates to empower the estimation of vibrational energies of compound securities in surface upgraded Raman spectroscopy. Gold nanoparticles are very thick, consequently enabling them to be utilized as tests for transmission electron microscopy [12]. Gold nanoparticles can be readily dispersed, functionalized and are bio inert in nature. These particles have high X-ray attenuation power [13]. Gold nanoparticles have been generally utilized as a part of the field of radiation solution in radiation treatment due to the effective and focused on medication to the tumor site. The biotemplates utilized for the synthesis of gold nanoparticles provide excellent chemical stability and biocompatibility. They play a key role in preventing the agglomeration of gold nanoparticles. In our study, we have elucidated the antimicrobial application of synthesized gold nanopartilces against Candida albicans. It is a common
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human fungal pathogen ion exists as risk-free commensal which causes mucosal, cutaneous and systemic infection in immune compromised individuals or patients undergoing long-term treatment with antibiotics. The incidence of invasive fungal infections is on the rise due to the increased use of immunosuppressive procedures and invasive health checkup measures. Side effects of the present treatment regimens and the emergence of multi-drug resistance (MDR) are problems affecting efficient therapy against infections caused by C. albicans[14]. Therefore, it is an urgent need to search alternative agents for the treatment of Candida infections. In this study we have focused on green synthesis of gold nanoparticles as it is ecofriendly. This synthesis was carried out in aqueous medium under microwave using starch as a template cum stabilizing agent [15]. These particles are characterized by UV visible spectroscopy, Transmission Electron Microscopy and Atomic force microscopy [16]. The antimicrobial efficiency of gold nanoparticles was tested against Candida albicans.
2. EXPERIMENTAL SECTION 2.1 Materials:
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In this method we used chemicals such as chloro auric acid (HAuCl4) and freshly prepared starch solution in aqueous medium. Chloro auric acid and starch purchased from Sigma Aldrich and Merck respectively were of analytical grade. All media chemicals YEPD (yeast extract peptone dextrose), sodium chloride and agar were purchased from HI Media (Mumbai, India).The chemicals were used without any further purification.
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2.2 Synthesis of gold nanoparticles using starch as bio-template
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Gold nanoparticles were synthesized by the reduction of Au3+ (HAuCl4) to Au0. In our study, we adopted the green synthetic method which is environmentally benign. 2% aqueous starch solution was prepared by taking 2g starch in 100ml of distilled water. A small quantity of HAuCl4 solution (1 mM) was added to the starch solution. The reaction mixture was stirred vigorously for about 30 minutes. The solution so obtained was irradiated with microwaves for about 3 minutes. The colour change from yellow to wine red confirmed the formation of gold nanoparticles. The outcome of microwave assisted synthesis is the formation of gold nanoparticles with uniform size and shape on starch template. 2.3 Characterization of
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Gold nanoparticles on starch template 2.3.1 UV-visible absorption spectroscopy Formation of gold nanoparticles on starch as bio-template was tested and confirmed by the UV-visible absorption spectroscopy. The absorption peak was studied by employing (Spectrophotometer model) using distilled water as reference sample. The sample (1 mM) was analyzed in the wavelength range of 200 nm - 800 nm.
2.3.2. X-ray Diffraction
Powder X-ray diffraction (XRD) patterns of the nanoparticles were collected at room temperature using an XPERT PRO,PANalytical instrument equipped with graphite monochromatized Cu-Ka radiation as the X-ray source. JCPDS database was used for phase identification of the synthesized gold nanoparticles.
2.3.3 Transmission electron spectroscopy Transmission Electron Microscopy were performed on a JEOL 3010 instrument operated at an accelerating voltage of 300 kV. Samples for TEM were prepared by ultrasonically dispersing the dried samples in water. A drop of well sonicated nanoparticle solution were put on a copper grid (300 mesh) with a layer of amorphous carbon kept on filter paper and was air dried. After 30 minutes, the grid was gently tapped to get rid of excess of water.
2.3.4 Atomic force spectroscopy
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AFM studies were carried out using Agilent technologies, 5500 Pico plus AFM system. All the images were taken using aquastic mode with cantilevers having resonance frequency 150 -300 kHz, tip height 10-15 μm, Mica was preferred as a solid substrate and used immediately after the cleavage in clean atmosphere. During this characterization process, the probe and cantilever were immersed completely in water solution. 2.4 Broth micro dilution assay
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MIC was determined by broth micro-dilution method as described in method M27-A3 from the Clinical and Laboratory Standards Institute (CLSI) formerly NCCLS [17]. Briefly, 100 𝜇L of media was placed at each well of the 96-well plate following the addition of the gold nanoparticle with the remaining media and then was serially diluted. 100 𝜇L of cell suspension (in normal saline to an OD6000.1) was added to each well of the plate and OD600was measured after 48 hours at 30 oC. The MIC80 was defined as the concentration at which the 80% of the growth was inhibited .
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2.3.5 Spot assay Spot assays were performed using a previously described method [18]. Briefly, for the spot assay 5 𝜇L of fivefold serial dilutions of each yeast culture (each with cells suspended in normal saline to an OD600nm of 0.1) was spotted onto YEPD plates in the absence (control) and in presence of gold nanoparticle.. Growth difference was measured after incubation at 30oC for 48 hours. The concentrations used in this study are specified in figure legends.
3. Result and Discussion 3.1. UV Visible spectroscopy Gold nanoparticles are attention-grabbing due to their high chemical stability, localized surface plasmon resonance (SPR), high catalytic activity and conductivity [19]. SPR is a measure of resonant oscillation of conduction electrons at the interface between negative and positive permittivity material stimulated by incident light. Figure 1 shows the UV-visible
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absorption spectrum of gold nanoparticles around 530 nm. UV-vis spectrum of gold nanoparticles at 530 nm confirms the formation of gold nanoparticles by reduction of Au3+ to Au0. The spectrum clearly shows the gold nanoparticles synthesized on starch template provide good chemical stability. The Surface Plasmon Resonance wavelength of gold nanoparticles gives a narrow peak which further confirms the formation of gold nanoparticles with lower particle size distribution [20].
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3.2 X-ray Diffraction
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Fig 1: SPR of gold nanoparticles
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The crystallinity and Structural characterization of the synthesized gold nanoparticles are performed using XRD analysis. XRD pattern of the synthesized gold nanoparticles are shown in Figure 2. There are mainly four peaks appeared in the XRD pattern. The characteristic peaks corresponding to (111), (200), (220) and (311 of u are located at . . . 77.30o respectively. The result indicates that the sample is composed of crystalline gold [21].
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Fig. 2 XRD of gold nanoparticle
3.3 Transmission electron microscopy
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TEM analysis was carried out to study the morphology of the gold nanoparticles using starch template. The TEM image of Gold nanoparticles is displayed in Figure 3. Analysis of the gold nanoparticles by means of TEM evidenced the formation of spherical nanoparticles with a particle size distribution in the range of less than 10 nm. The TEM image features the monodispersed gold nanoparticles synthesized on starch template. TEM images shows the non-aggregated gold nanoparticles with good colloidal stability; thus corroborating starch as a potential reducing and stabilizing agent [22]. The high resolution TEM images of given nanoparticles as shown in fig. 4 (a) & 4 (b) at 5 nm scale and 50 nm scale respectively.
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Fig.3.TEM images of synthesized gold nanoparticles (a) at 5 nm scale & (b) at 50 nm scale
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. Fig. (a HRTEM image of synthesized gold nanoparticles with space between atoms is . nm and (b S ED pattern of synthesized gold nanoparticles.
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The HRTEM image (a of synthesized gold nanoparticles with interatomic spacing of . nm further confirms the formation of gold nanoparticles. Figure (b shows the S ED pattern of synthesized gold nanoparticles [ ]. The S ED image displays the diffraction rings signifying each of the crystal plane and also indicate the formation of very small sized nanoparticles (< 1 nm . The bright spots on the diffraction pattern confirm the crystallinity of the gold nanoparticles synthesized using starch template.
3.4 Atomic force microscopy AFM results present the topography and surface roughness of gold nanoparticles synthesized on starch template. The figure 5(b) and 5(c) are the topographic images while 5(bʹ and 5(cʹ
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represent the 3D images of the surface of gold nanoparticles on starch template [24]. The AFM images show the surface uniformity signifying the formation of gold nanoparticles with uniform size and shape. The nanoparticles synthesized are found to be very small with particle size around 10nm.
topographic images and
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Fig. 5 FM images of synthesized of gold nanoparticles :(b b’ (c c’ three dimensional images of respectively.
3.5 Gold nanoparticles are efficient inhibitor of C. albicans
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The gold nanoparticles affect the H+-ATPase mediated H+ pumping of various Candida species and also show admirable antimicobial activity [25]. To find out the antifungal activity of gold nanoparticle against C. albicans, we have performed two independent methods of susceptibility assays namely minimum inhibitory concentration (MIC80) using broth microdilution method and spot assay. We observed that gold nanoparticles were efficient to inhibit the growth of human pathogen, C. albicans. This was evident from broth micro dilution assay which depicts that MIC of gold nanoparticles was found to be 0.5 mM (Fig. 5). We further substantiate our findings by spot assay which confirms that 0.5 mM of gold nanoparticles were sufficient to elude the growth of fungal cells (Fig. 6). The pronounced antimicrobial activity is ascribed to the small size and large surface area of gold nanoparticles. As the particle size of the nanoparticles decreases the surface area increases. The increased surface area results in the enhanced interaction of gold nanoparticles with the C. albican resulting in the improved antifungal activity. Smaller the particle size, greater is the ability of nanoparticles to enter the microbial cell and interfere with the regular cell metabolism [26, 27]. . Considering the fact that C. albicans is developing resistance against currently available antifungal drugs, combinatorial therapy using these nanoparticles could be an alternative approach that could be adopted to boost the effectiveness of these drugs.
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Fig.6 Minimum inhibitory concentrations (MICs) of Candida albicans SC5314 was determined by broth microdilution in the presence of gold nanoparticle, Arrow depicted the MIC of gold nanoparticles against Candida albicans at 0.5 mM.
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(b)
Fig.7 Spot assay of C. albicans SC5314 in the absence (control) (a) and presence of Gold
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nanoparticles (b).
The gold nanoparticles are synthesized on starch template under microwave assisted method. The starch provides a good platform for the synthesis of gold nanoparticles, where it acts as both reducing cum stabilizing agent [28]. The concentration of gold precursor and starch composition varied simultaneous and found that the optimal concentration for the gold nanoparticle is 1 mM gold precursor in 2% starch solution. The presence of hydroxyl group in starch template provides active sites for nucleation and growth of nanoparticles without any agglomeration of the nanoparticles. The synthesized nanoparticles are found to very uniform and monodisperse in nature [29]. Particle size distribution also found to be very
small which is confirmed from TEM results. The nanoparticles are biocompatible and the presence of hydroxyl group in starch helps the reduction of Au3+ to Au0. The synthesized nanoparticles are found to be very efficient in inhibiting the growth of human fungal pathogen Candida albicans.
4. Conclusion
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In this study, we have adopted simple efficient and economically “green” approach for the synthesis of Gold nanoparticles. The syntheses of gold nanoparticles were carried out using microwave assisted synthesis using starch as template in aqueous medium. The bio-template starch significantly plays the dual role of being the reducing agent as well as stabilizing agent. This mode of synthesizing nanoparticles is advantageous over other reduction methods as it does not involve any harmful organic templates. The formation of crystalline, non-aggregated, spherical and monodispersed nanoparticles were confirmed by TEM analysis. AFM results being in good rapport with the TEM analysis confirms the uniform particle size distribution over the starch template. Also the gold nanoparticles synthesized over the starch template were successful in inhibiting the growth of Candida albicans. The MIC was found to be 0.5 mM which was concurrent with the spot assay.
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Acknoledgement
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The Authors thank Centre for Research, CHRIST (Deemed to be University) for the financial support through Major Research Project (MRP - 1830). Authors also thank Amity University Haryana for the various characterization and application studies.
References [1]. A.Lalitha, R.Subbaiya*and P.Ponmurugan, Int.J.Curr.Microbiol.App.Sci (2013) 2(6): 228-235 [2]. G.Parthasarathy, M. Saroja, M. Venkatachalam, P.Gowthaman & V. K. Evanjelene, Imperial Journal of Interdisciplinary Research (IJIR), 2, 2016, 1362-2454. [3]C. N. RamachandraRao, Giridhar U. Kulkarni, P. John Thomasan Peter P. Edwards, Chem. Soc. Rev., 2000, 29, 27–35. [4] S.Zeng; Yong, Ken-Tye; Roy, Indrajit; Dinh, Xuan-Quyen; Yu, Xia; Luan, Feng; et al.(2001) Plasmonics. 6 (3): 491–506. doi:10.1007/s11468-011-9228-1.
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[5]Sharma, Vivek; Park, Kyoungweon; Srinivasarao, Mohan (2009). Material Science and Engineering Reports. 65 (1–3): 1–38. doi:10.1016/j.mser.2009.02.002. [6]Freestone, Ian; Meeks, Nigel; Sax, Margaret; Higgitt, Catherine. . Gold Bulletin. 40 (4): 270–277. doi:10.1007/BF03215599. ISSN 0017-1557.
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[7]G.Dodson ,A.Wlodawer,Trends Biochem Sci.23(9) (1998) 347-352.
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[8]Crewe, Albert V; Isaacson, M. and Johnson, D.; Johnson, D. (1969). Rev. Sci. Inst. 40 (2): 241–246. Bibcode:1969RScI...40..241C. doi:10.1063/1.1683910. J.,
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[9]Crewe, Albert V; Wall, J. and Langmore, (1970).. doi:10.1126/science.168.3937.1338. PMID 17731040.
[10]S.R.Saptarshi, A.Duschlz,A.L.Lopatal,J.Nanobiotechnol.11(26) (2013)1-12
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[11] Fultz, B & Howe, J (2007). . Springer. ISBN 3-540-73885-1.
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[12] Murphy, Douglas B. (2002). Fundamentals of Light Microscopy and Electronic Imaging. New York: John Wiley & Sons. ISBN 9780471234296.
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[13]Champness, P. E. (2001. Garland Science. ISBN 978-1859961476. [14]Hubbard, A (1995). . CRC Press. ISBN 0-8493-8911-9.
[15]. Hans S, Fatima Z, Hameed S. J Glob Antimicrob Resist. 2019 Jan 16;17:263-275. doi: 10.1016/j.jgar.2019.01.011.
[16][Egerton, R (2005). Physical principles of electron microscopy. Springer. ISBN 0-38725800-0.
[17] Huang, D.; Liao, F.; Molesa, S.; Redinger, D.; Subramanian, V. Journal of the Electrochemical Society, 2003, 150, G412-417. [18] Stuchinskaya, T.; Moreno, M.; Cook, M. J.; Edwards, D. R.; Russell, D. A. Photochem. Photobiol. Sci., 2011, 10, 822-831 [19]Brown, S. D.; Nativo, P.; Smith, J.-A.; Stirling, D.; Edwards, P. R.; Venugopal, B.; Flint, D. J.; Plumb, J. A.; Graham, D.; Wheate, N. J. J. Am. Chem. Soc., 2010, 132, 4678-4684 [20] Binnig, G.; Quate, C. F.; Gerber, C. (1986). Physical Review Letters. 56: 930–933. .
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[21] H. Hinterwirth, W. Lindner, M. Lämmerhofer, Anal. Chim. Acta 733 (2012) 90–97. [22]. Ahmad T, Wani IA, Manzoor N, Ahmed J, Asiri AM. Colloids Surf Biointerfaces. 2013
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[23]. Wani IA, Ahmad T, Manzoor N. Size and shape dependant antifungal activity of gold nanoparticles: a case study of Candida. Colloids Surf B Biointerfaces. 2013 Jan 1;101:162-
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[24]. Tokeer Ahmad, Irshad A. Wani, Irfan H. Lone , Aparna Ganguly, Nikhat Manzoor , Aijaz Ahmad, Jahangeer Ahmed, Ayed S. Al-Shihri, Materials Research Bulletin 48 (2013) 12–20
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[25] Cappella, B; Dietler, G (1999). Surface Science Reports. 34 (1–3): 1–104 [26]M. Nidhin, R. Indumathy, K.J. Sreeram, B.U. Nair, Bull. Mater. Sci. 31 (2008) 93–96.
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[27] S.M. Kelly, T.J. Jess, N.C. Price, Biochim. Biophys. Acta 1751 (2005) 119–139
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[28] S. Majumder, S. Khamrui, R. Banerjee, P. Bhowmik, U. Sen, Biochim. Biophys. Acta 1854 (1) (2015) 55–64.
[29] Lang, K.M.; D. A. Hite; R. W. Simmonds; R. McDermott; D. P. Pappas; John M. Martinis (2004. 75 (8): 2726–2731.
S0025-5408(18)32533-9 https://doi.org/10.1016/j.materresbull.2019.110563 110563
Reference:
MRB 110563
To appear in:
MRB
Received date: Revised date: Accepted date:
17 September 2018 20 July 2019 25 July 2019
Please cite this article as: M N, D S, Hans S, Varghese A, Fatima Z, Hameed S, Studies on the antifungal activity of biotemplated gold nanoparticles over Candida albicans, Materials Research Bulletin (2019), https://doi.org/10.1016/j.materresbull.2019.110563 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Studies on the antifungal activity of biotemplated gold nanoparticles over Candida albicans Nidhin Ma,*, Saneha Db, Sandeep Hansc, Anitha Varghesea, Zeeshan Fatimac, Saif Hameedc,* a
Department of Chemistry, CHRIST (Deemed to be University), Bengaluru, Karnataka 560029, India Department of Chemistry, Amity School of Applied Sciences, Amity University Haryana Amity Education Valley, Gurgaon, Haryana 122413,India c Amity Institute of Biotechnology, Amity University Haryana, Amity Education Valley, Gurgaon, Haryana 122413,India b
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*Corresponding Authors: Nidhin M, Department of Chemistry, CHRIST (Deemed to be University), Bengaluru, Karnataka 560029
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Email: [email protected] , [email protected]
122413.
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Email : [email protected]
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Dr. Saif Hameed, Amity Institute of Biotechnology, Amity University Haryana, Gurgaon-
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Graphical abstract
Highlights The synthesis of gold nanoparticles and their biomedical applications are always a very hot topic in the scientific community. This paper highlights the synthesis of gold nanoparticles of uniform size around 5nm with spherical shape using starch as a biotemplate. X-Ray diffraction, UV-visible Spectroscopy, Transmission Electron Microscopy and Atomic Force Microscopy were employed to characterize or analyzed the synthesized gold nanoparticles.
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The biomedical applications of the synthesized gold nanoparticles were carried out against most prevalent human fungal pathogen, Candida albicans. Broth micro dilution assay to determine Minimum inhibitory concentration (MIC) was used to find out the concentration of
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gold nanoparticles at which it inhibits the growth of fungal cells. We observed that 0. 5 mM
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concentration was sufficient to inhibit the growth of cells in which was also confirmed by
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spot assay.
ABSTRACT Green synthesis and applications of gold nanoparticles are more fascinating research area due to their unique optical properties and high X-ray attenuation power. In this study, we have synthesized gold nanoparticles of uniform size (5 nm) with spherical shape. UV-visible spectroscopy, Transmission Electron Microscopy and Atomic Force Microscopy were employed to characterize the synthesized gold nanoparticles. The biomedical applications of the synthesized gold nanoparticles were carried out against most prevalent human fungal pathogen, Candida albicans. Broth micro dilution assay was used to determine minimum inhibitory concentration (MIC). We observed that 0. 5 mM concentration was effective in inhibiting the growth of fungal cells which was later confirmed by spot assay. Key Words: Gold Nanoparticles, Antifungal Activity, Candida albicans, Micro Dilution
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Assay, Green Synthesis
1. Introduction
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In the modern era of material science, nanoparticles have become the centre of attraction to the scientific community. In current years, nanotechnology is one of the most researched areas due to their noticeable performance in electronics, optics and photonics. Nanoparticles are the fundamental structures of nanotechnology.[1] Nanoscience and technology is an interdisciplinary broad area of research and development activity that has been growing dynamically worldwide in the past few years. Nanoparticles are the simplest form of structures with the range of 1-100 nm [2]. Nanoparticles have different physical and chemical properties such as higher surface area, mechanical strength and high reactivity. Gold nanoparticles are unique in optical property as gold is yellow in shading, strong in state where as gold nanoparticles are wine red shading arrangement against oxidant [3]. Gold nanoparticles display different sizes extending from 1 nm to 8 μm and they likewise show distinctive shapes, such as round, sub-octahedral, octahedral, decahedral, sporadic shape, tetrahedral, hexagonal platelets and nanorods. Among all these shapes, circular molded nanoparticles are most steady and show alluring optical properties when contrasted with the triangular formed nanoparticles [4]. Conventional physical and chemical methods are used to prepare metal nanoparticles from toxic chemicals [5]. Moreover, these methods are very expensive and not environmentally friendly [6]. Green synthesis of gold nanoparticles using various templates such as polysaccharides, fungi and plant extracts are found to be environmentally benign [7]. These attractive green strategies are free from toxic chemicals and toxic materials. Gold nanoparticle synthesized from bio templates offers a route for large scale production of different metallic nanoparticles [8].
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Bio templated gold nanoparticles are utilized to detect cancer cells. Increased surface area of gold nanoparticles in solution which contribute to their enhanced physio-chemical properties which are useful in a variety of fields such as antimicrobial agents [9], bio-molecular detection, diagnostics, catalysis, biomedical and bio sensing devices. Gold nanoparticles are utilized as efficient materials for water purification [10]. These are also used in interface resistors, conductors and different components of an electronic chip. In photodynamic treatment, when light is connected to a tumor containing gold nanoparticles, the particles quickly warm up, executing tumor cells [11]. Gold nanoparticles are used as substrates to empower the estimation of vibrational energies of compound securities in surface upgraded Raman spectroscopy. Gold nanoparticles are very thick, consequently enabling them to be utilized as tests for transmission electron microscopy [12]. Gold nanoparticles can be readily dispersed, functionalized and are bio inert in nature. These particles have high X-ray attenuation power [13]. Gold nanoparticles have been generally utilized as a part of the field of radiation solution in radiation treatment due to the effective and focused on medication to the tumor site. The biotemplates utilized for the synthesis of gold nanoparticles provide excellent chemical stability and biocompatibility. They play a key role in preventing the agglomeration of gold nanoparticles. In our study, we have elucidated the antimicrobial application of synthesized gold nanopartilces against Candida albicans. It is a common
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human fungal pathogen ion exists as risk-free commensal which causes mucosal, cutaneous and systemic infection in immune compromised individuals or patients undergoing long-term treatment with antibiotics. The incidence of invasive fungal infections is on the rise due to the increased use of immunosuppressive procedures and invasive health checkup measures. Side effects of the present treatment regimens and the emergence of multi-drug resistance (MDR) are problems affecting efficient therapy against infections caused by C. albicans[14]. Therefore, it is an urgent need to search alternative agents for the treatment of Candida infections. In this study we have focused on green synthesis of gold nanoparticles as it is ecofriendly. This synthesis was carried out in aqueous medium under microwave using starch as a template cum stabilizing agent [15]. These particles are characterized by UV visible spectroscopy, Transmission Electron Microscopy and Atomic force microscopy [16]. The antimicrobial efficiency of gold nanoparticles was tested against Candida albicans.
2. EXPERIMENTAL SECTION 2.1 Materials:
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In this method we used chemicals such as chloro auric acid (HAuCl4) and freshly prepared starch solution in aqueous medium. Chloro auric acid and starch purchased from Sigma Aldrich and Merck respectively were of analytical grade. All media chemicals YEPD (yeast extract peptone dextrose), sodium chloride and agar were purchased from HI Media (Mumbai, India).The chemicals were used without any further purification.
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2.2 Synthesis of gold nanoparticles using starch as bio-template
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Gold nanoparticles were synthesized by the reduction of Au3+ (HAuCl4) to Au0. In our study, we adopted the green synthetic method which is environmentally benign. 2% aqueous starch solution was prepared by taking 2g starch in 100ml of distilled water. A small quantity of HAuCl4 solution (1 mM) was added to the starch solution. The reaction mixture was stirred vigorously for about 30 minutes. The solution so obtained was irradiated with microwaves for about 3 minutes. The colour change from yellow to wine red confirmed the formation of gold nanoparticles. The outcome of microwave assisted synthesis is the formation of gold nanoparticles with uniform size and shape on starch template. 2.3 Characterization of
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Gold nanoparticles on starch template 2.3.1 UV-visible absorption spectroscopy Formation of gold nanoparticles on starch as bio-template was tested and confirmed by the UV-visible absorption spectroscopy. The absorption peak was studied by employing (Spectrophotometer model) using distilled water as reference sample. The sample (1 mM) was analyzed in the wavelength range of 200 nm - 800 nm.
2.3.2. X-ray Diffraction
Powder X-ray diffraction (XRD) patterns of the nanoparticles were collected at room temperature using an XPERT PRO,PANalytical instrument equipped with graphite monochromatized Cu-Ka radiation as the X-ray source. JCPDS database was used for phase identification of the synthesized gold nanoparticles.
2.3.3 Transmission electron spectroscopy Transmission Electron Microscopy were performed on a JEOL 3010 instrument operated at an accelerating voltage of 300 kV. Samples for TEM were prepared by ultrasonically dispersing the dried samples in water. A drop of well sonicated nanoparticle solution were put on a copper grid (300 mesh) with a layer of amorphous carbon kept on filter paper and was air dried. After 30 minutes, the grid was gently tapped to get rid of excess of water.
2.3.4 Atomic force spectroscopy
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AFM studies were carried out using Agilent technologies, 5500 Pico plus AFM system. All the images were taken using aquastic mode with cantilevers having resonance frequency 150 -300 kHz, tip height 10-15 μm, Mica was preferred as a solid substrate and used immediately after the cleavage in clean atmosphere. During this characterization process, the probe and cantilever were immersed completely in water solution. 2.4 Broth micro dilution assay
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MIC was determined by broth micro-dilution method as described in method M27-A3 from the Clinical and Laboratory Standards Institute (CLSI) formerly NCCLS [17]. Briefly, 100 𝜇L of media was placed at each well of the 96-well plate following the addition of the gold nanoparticle with the remaining media and then was serially diluted. 100 𝜇L of cell suspension (in normal saline to an OD6000.1) was added to each well of the plate and OD600was measured after 48 hours at 30 oC. The MIC80 was defined as the concentration at which the 80% of the growth was inhibited .
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2.3.5 Spot assay Spot assays were performed using a previously described method [18]. Briefly, for the spot assay 5 𝜇L of fivefold serial dilutions of each yeast culture (each with cells suspended in normal saline to an OD600nm of 0.1) was spotted onto YEPD plates in the absence (control) and in presence of gold nanoparticle.. Growth difference was measured after incubation at 30oC for 48 hours. The concentrations used in this study are specified in figure legends.
3. Result and Discussion 3.1. UV Visible spectroscopy Gold nanoparticles are attention-grabbing due to their high chemical stability, localized surface plasmon resonance (SPR), high catalytic activity and conductivity [19]. SPR is a measure of resonant oscillation of conduction electrons at the interface between negative and positive permittivity material stimulated by incident light. Figure 1 shows the UV-visible
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absorption spectrum of gold nanoparticles around 530 nm. UV-vis spectrum of gold nanoparticles at 530 nm confirms the formation of gold nanoparticles by reduction of Au3+ to Au0. The spectrum clearly shows the gold nanoparticles synthesized on starch template provide good chemical stability. The Surface Plasmon Resonance wavelength of gold nanoparticles gives a narrow peak which further confirms the formation of gold nanoparticles with lower particle size distribution [20].
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3.2 X-ray Diffraction
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Fig 1: SPR of gold nanoparticles
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The crystallinity and Structural characterization of the synthesized gold nanoparticles are performed using XRD analysis. XRD pattern of the synthesized gold nanoparticles are shown in Figure 2. There are mainly four peaks appeared in the XRD pattern. The characteristic peaks corresponding to (111), (200), (220) and (311 of u are located at . . . 77.30o respectively. The result indicates that the sample is composed of crystalline gold [21].
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Fig. 2 XRD of gold nanoparticle
3.3 Transmission electron microscopy
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TEM analysis was carried out to study the morphology of the gold nanoparticles using starch template. The TEM image of Gold nanoparticles is displayed in Figure 3. Analysis of the gold nanoparticles by means of TEM evidenced the formation of spherical nanoparticles with a particle size distribution in the range of less than 10 nm. The TEM image features the monodispersed gold nanoparticles synthesized on starch template. TEM images shows the non-aggregated gold nanoparticles with good colloidal stability; thus corroborating starch as a potential reducing and stabilizing agent [22]. The high resolution TEM images of given nanoparticles as shown in fig. 4 (a) & 4 (b) at 5 nm scale and 50 nm scale respectively.
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Fig.3.TEM images of synthesized gold nanoparticles (a) at 5 nm scale & (b) at 50 nm scale
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. Fig. (a HRTEM image of synthesized gold nanoparticles with space between atoms is . nm and (b S ED pattern of synthesized gold nanoparticles.
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The HRTEM image (a of synthesized gold nanoparticles with interatomic spacing of . nm further confirms the formation of gold nanoparticles. Figure (b shows the S ED pattern of synthesized gold nanoparticles [ ]. The S ED image displays the diffraction rings signifying each of the crystal plane and also indicate the formation of very small sized nanoparticles (< 1 nm . The bright spots on the diffraction pattern confirm the crystallinity of the gold nanoparticles synthesized using starch template.
3.4 Atomic force microscopy AFM results present the topography and surface roughness of gold nanoparticles synthesized on starch template. The figure 5(b) and 5(c) are the topographic images while 5(bʹ and 5(cʹ
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represent the 3D images of the surface of gold nanoparticles on starch template [24]. The AFM images show the surface uniformity signifying the formation of gold nanoparticles with uniform size and shape. The nanoparticles synthesized are found to be very small with particle size around 10nm.
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Fig. 5 FM images of synthesized of gold nanoparticles :(b b’ (c c’ three dimensional images of respectively.
3.5 Gold nanoparticles are efficient inhibitor of C. albicans
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The gold nanoparticles affect the H+-ATPase mediated H+ pumping of various Candida species and also show admirable antimicobial activity [25]. To find out the antifungal activity of gold nanoparticle against C. albicans, we have performed two independent methods of susceptibility assays namely minimum inhibitory concentration (MIC80) using broth microdilution method and spot assay. We observed that gold nanoparticles were efficient to inhibit the growth of human pathogen, C. albicans. This was evident from broth micro dilution assay which depicts that MIC of gold nanoparticles was found to be 0.5 mM (Fig. 5). We further substantiate our findings by spot assay which confirms that 0.5 mM of gold nanoparticles were sufficient to elude the growth of fungal cells (Fig. 6). The pronounced antimicrobial activity is ascribed to the small size and large surface area of gold nanoparticles. As the particle size of the nanoparticles decreases the surface area increases. The increased surface area results in the enhanced interaction of gold nanoparticles with the C. albican resulting in the improved antifungal activity. Smaller the particle size, greater is the ability of nanoparticles to enter the microbial cell and interfere with the regular cell metabolism [26, 27]. . Considering the fact that C. albicans is developing resistance against currently available antifungal drugs, combinatorial therapy using these nanoparticles could be an alternative approach that could be adopted to boost the effectiveness of these drugs.
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Fig.6 Minimum inhibitory concentrations (MICs) of Candida albicans SC5314 was determined by broth microdilution in the presence of gold nanoparticle, Arrow depicted the MIC of gold nanoparticles against Candida albicans at 0.5 mM.
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(b)
Fig.7 Spot assay of C. albicans SC5314 in the absence (control) (a) and presence of Gold
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nanoparticles (b).
The gold nanoparticles are synthesized on starch template under microwave assisted method. The starch provides a good platform for the synthesis of gold nanoparticles, where it acts as both reducing cum stabilizing agent [28]. The concentration of gold precursor and starch composition varied simultaneous and found that the optimal concentration for the gold nanoparticle is 1 mM gold precursor in 2% starch solution. The presence of hydroxyl group in starch template provides active sites for nucleation and growth of nanoparticles without any agglomeration of the nanoparticles. The synthesized nanoparticles are found to very uniform and monodisperse in nature [29]. Particle size distribution also found to be very
small which is confirmed from TEM results. The nanoparticles are biocompatible and the presence of hydroxyl group in starch helps the reduction of Au3+ to Au0. The synthesized nanoparticles are found to be very efficient in inhibiting the growth of human fungal pathogen Candida albicans.
4. Conclusion
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In this study, we have adopted simple efficient and economically “green” approach for the synthesis of Gold nanoparticles. The syntheses of gold nanoparticles were carried out using microwave assisted synthesis using starch as template in aqueous medium. The bio-template starch significantly plays the dual role of being the reducing agent as well as stabilizing agent. This mode of synthesizing nanoparticles is advantageous over other reduction methods as it does not involve any harmful organic templates. The formation of crystalline, non-aggregated, spherical and monodispersed nanoparticles were confirmed by TEM analysis. AFM results being in good rapport with the TEM analysis confirms the uniform particle size distribution over the starch template. Also the gold nanoparticles synthesized over the starch template were successful in inhibiting the growth of Candida albicans. The MIC was found to be 0.5 mM which was concurrent with the spot assay.
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Acknoledgement
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The Authors thank Centre for Research, CHRIST (Deemed to be University) for the financial support through Major Research Project (MRP - 1830). Authors also thank Amity University Haryana for the various characterization and application studies.
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